Background: Insulin-degrading enzyme (IDE) is the best characterized catabolic enzyme implicated in insulin proteolysis. Results: Newly discovered dual exosite IDE inhibitors do not significantly affect insulin action or clearance. Conclusion: IDE catabolism does not appear to be the primary mechanism of insulin clearance in vivo. Significance: These IDE inhibitors will enable broader investigation of IDE function.

The first potent inhibitors of glutamate racemase (MurI) enzyme that show whole cell antibacterial activity are described. Optically pure 4-substituted D-glutamic acid analogues with (2R,4S) stereochemistry and bearing aryl-, heteroaryl-, cinnamyl-, or biaryl-methyl substituents represent a novel class of glutamate racemase inhibitors. Exploration of the D-Glu core led to the identification of lead compounds (-)-8 and 10. 2-Naphthylmethyl derivative 10 was found to be a potent competitive inhibitor of glutamate racemase activity (K(i) = 16 nM, circular dichroism assay; IC(50) = 0.1 microg/mL high-performance liquid chromatography (HPLC) assay). Thorough structure-activity relationship (SAR) studies led to benzothienyl derivatives such as 69 and 74 with increased potency (IC(50) = 0.036 and 0.01 microg/mL, respectively, HPLC assay). These compounds showed potent whole cell antibacterial activity against S. pneumoniae PN-R6, and good correlation with the enzyme assay. Compounds 69, 74 and biaryl derivative 52 showed efficacy in an in vivo murine thigh infection model against Streptococcus pneumoniae. Data described herein suggest that glutamate racemase may be a viable target for developing new antibacterial agents.

Studies on the mechanism of leukotriene B4 biosynthesis in suspensions composed of neutrophils plus erythrocytes indicate that human erythrocytes convert neutrophil-derived leukotriene A4 into leukotriene B4. Leukotriene B4 formation by neutrophils in the presence of erythrocytes exceeded that from corresponding suspensions of neutrophils alone. The increase was proportional to the erythrocyte content of the suspension. The erythrocyte-dependent increase in leukotriene B4 biosynthesis did not equal the arithmetic sum of calcium ionophore-dependent biosynthesis by neutrophils plus calcium ionophore-dependent biosynthesis by erythrocytes, since erythrocytes produced no leukotriene B4 upon incubation with ionophore A23187. Erythrocytes did not stimulate 5-lipoxygenase activity within neutrophils, since the erythrocyte effect was confined to enzymatic hydration: leukotriene B4 increased coincident with decreased formation of 5,12-dihydroxyicosatetraenoic acids derived from nonenzymatic hydration. Biosynthesis of leukotriene B4 within the erythrocyte, from neutrophil-derived leukotriene A4, was established by comparing the effect of normal erythrocytes with erythrocytes containing a leukotriene A4 hydrolase that was inactivated by the substrate. In the latter case, leukotriene B4 formation increased by only 30-40%; in the former case, it increased by 100-200%. Transcellular biosynthesis of leukotriene B4 from erythrocyte-neutrophil interactions (i) explains the paradoxical presence of leukotriene A4 hydrolase within erythrocytes, a cell incapable of synthesizing leukotriene A4; (u) affords a mechanism to overcome rate limitations or "suicide inactivation" of leukotriene A4 hydrolase in neutrophils; (iii) exploits a cryptic capacity within erythrocytes, provisionally dormant cells in terms of icosanoid biosynthesis; (iv) indicates that the biosynthetic capacity of cell combinations is not necessarily equivalent to the sum of their separate capacities.Enzymatic cooperation between separate cell types may be a significant, but neglected, mechanism for the biosynthesis of certain prostaglandins or leukotrienes. This concept originated from studies on platelet-endothelial cell interactions, which showed that prostaglandin I2 (PGI2), an antithrombotic icosanoid, was produced by endothelial cells from plateletderived prostaglandin endoperoxide H2 (1-5). Analogous transcellular biosynthetic processes are conceivable for other cell combinations and for other arachidonic acid metabolites; however, few biologically relevant examples exist. Requirements for this process include "acceptor" cells with a constitutive enzymatic capacity for the formation of a biologically active icosanoid product but a deficient, or disabled, capacity for substrate generation, plus auxiliary "donor" cells that can fulfill that corresponding substrate requirement. In this context, neutrophil-erythrocyte interactions attracted our attention as potential sources for transcellular biosynthesis of leukotrienes (6).Ordinarily, biosynthesis of leu...

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a “testing funnel” of assays to identify genuine hits using high‐throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

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We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC of 24nM for inhibition of PGE formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC in both rat (5mg/kg) and dog (3mg/kg) for over twelve hours.